Abstract

Two substrates of insulin-degrading enzyme (IDE), amyloid beta-protein (Abeta) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Abeta levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Abeta and insulin degradation in vivo and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE --). IDE deficiency resulted in a >50% decrease in Abeta degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE -- mice showed increased cerebral accumulation of endogenous Abeta, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the beta-amyloid precursor protein, which was recently found to be degraded by IDE in vitro. Together with emerging genetic evidence, our in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD.

Aβ degradation and accumulation in IDE gene-disrupted mice. (A) Aβ degradation in brain membrane fractions. 125I-Aβ1-40 proteolysis in Na2CO3-washed brain membranes from two IDE −/− and two WT (+/+) mice was measured by TCA precipitation assay in the presence or absence of unlabeled insulin (3-h time point shown). Bars represent means ± SEM of 4–10 determinations from two to three independent assays (*, P < 0.00001 by Student's t test for IDE −/− compared with the IDE +/+ without inhibitors). (B) Aβ degradation in primary neurons. Age-matched cortical neurons (7–22 days in vitro) from two IDE −/− (filled circles) and two +/+ (open squares) embryonic day 17 litters were incubated with 40 pM 125I-Aβ1-40, and TCA precipitation assays were performed at the indicated time points. Graph points represent the mean ± SEM of 9–11 determinations from five to six independent assays (*, P < 0.00001). Some error bars are obscured by the symbols for data points. (C) Endogenous Aβ levels in IDE −/− neuronal conditioned media. Cortical neurons from two IDE −/− and two +/+ embryonic day 17 litters were allowed to condition media for 7 (4–11 days in vitro) or 14 days (4–18 days in vitro) before being analyzed by Aβ X-40 and Aβ X-42 ELISA. X-40 and X-42 bars represent mean Aβ concentrations ± SEM of six to eight and four to six determinations, respectively, and each determination is a mean of duplicate ELISA values (*, P < 0.005; **, P = 0.01). (D) Endogenous Aβ levels in IDE −/− brains. Represented are the mean pmol of Aβ per g of brain ± SEM of six WT and seven −/− brains from 12-week-old animals determined by Aβ X-40 ELISA (*, P < 0.00001).

Cerebral levels of APP and AICD in IDE-deficient mice. Shown are immunoprecipitation immunoblots of brain lysate with antibody C8. Identical results were obtained with C8 immunoprecipitation followed by anti-0443 immunoblotting. (A) AICD in APP gene-disrupted (−/−), WT (+/+), and APP transgenic (Tg) mouse brains. Lane C60/Fe65, the positive control, is lysate from transfected COS cells coexpressing a 59-aa AICD construct, which runs slightly higher than the endogenous 50-aa AICD, and Fe65, which stabilizes AICD (28). (B) Full-length APP and AICD in IDE −/− vs. +/+ brain. The APP and AICD bands are from identical lanes of the same blot, but the APP bands are from a lighter exposure. (C) Dephosphorylation of AICD. After the final wash following immunoprecipitation, samples were incubated for 1 h with either calf intestinal phosphatase (CIP) or buffer alone.